Abstract

When DNA replication is slowed down, normally dormant replication origins are activated.
Recent work demonstrates that cells adapt by changing the organization of chromatin
loops and maintaining the new pattern of origin use in subsequent cell cycles.

Minireview

It is critical that chromosomal DNA is precisely duplicated during S phase of the
eukaryotic cell cycle, with no sections of DNA left unreplicated or replicated more
than once. There is a considerable plasticity in this process because cells license
many potential replication origins, of which only a small percentage are used in any
one cell cycle, with the others remaining 'dormant'. This means that the usage of
replication origins can change under different circumstances. For example, dormant
replication origins can be activated when replication forks are inhibited to allow
timely completion of the replication programme. A recent paper published in Nature by Courbet et al. [1] illustrates this plasticity of replication origin usage and shows that it is associated
with longer-term changes to the organization of chromatin loops. The changes to chromatin
organization can then directly affect the way that replication origins are used in
subsequent cell cycles.

Dormant origins and the plasticity of the replication program

The precise duplication of large eukaryotic chromosomes is a dauntingly complex task.
For the DNA to be completely replicated, replication forks need to be initiated at
thousands of replication origins scattered throughout the genome. This is made more
difficult by the fact that replication forks can frequently stall, for example if
they encounter damaged bases. It is also crucial that each replication origin does
not fire more than once in a single S phase, as this would lead to local amplification
of the DNA.

During late mitosis and early G1, the cell licenses replication origins for use in
the upcoming S phase by loading protein complexes composed of Mcm proteins (Mcm2-7
complexes) onto the origin DNA [2,3]. During S phase, Mcm2-7 at licensed origins can initiate replication forks. The Mcm2-7
complex moves with the replication forks, providing the essential DNA helicase activity
that unwinds the DNA. This means that when an origin initiates a pair of forks, it
is converted into the unlicensed state and cannot fire again. However, the mechanisms
that ensure the appropriate distribution and usage of replication origins on DNA are
poorly understood in animal cells. Many more origins are licensed in G1 than are actually
used, with around 90% of licensed origins being inefficient and remaining dormant
in any given S phase. When replication forks are stalled or slowed, dormant origins
are activated [4-6], which can help cells ensure complete genome replication [7-9].

Activation of dormant origins by a 'passive' mechanism

In their recent paper, Courbet et al. [1] investigated the regulation of origin usage in response to changes in replication
fork dynamics. Previous work from their lab had mapped a cluster of replication origins
in an amplified region surrounding the AMPD2 (adenosine monophosphate deaminase 2) locus in Chinese hamster fibroblasts [10]. Under conditions of normal fork movement, this region is predominantly replicated
from forks initiated at an origin termed oriGNAI3, though initiation was occasionally
observed at inefficient (dormant) origins termed oriA-oriF. When forks were slowed,
by reducing the cellular supply of deoxynucleotides, initiation at oriA-oriF was significantly
increased. This was associated with an overall increase in the number of initiation
events throughout the locus.

One simple explanation for these results is that origin firing is stochastic. Once
a genomic locus containing a cluster of origins becomes activated, dormant origins
within that cluster normally have only a brief time to fire before they are passively
replicated (and hence inactivated) by a fork from a neighboring origin. If replication
forks are slowed, dormant origins are more likely to fire simply because there is
an increased period of time before they are passively replicated. Consistent with
this 'passive' mechanism, it has been reported that under conditions of modest fork
slowing (when replication checkpoints are not strongly activated), there is a correlation
between the degree of fork slowing and the overall increase in origin density [1,8].

Long-term adaptation of origin usage

This passive activation of dormant origins is a rapid and transitory response that
should not affect the long-term behavior of cells. A key observation of Courbet et al. [1] is that in addition to this rapid response, cells also respond to changes in fork
dynamics by adapting origin usage in subsequent cell cycles. Cells were grown under
conditions in which forks could only move slowly, promoting a high rate of initiation
at oriA-oriF as well as at oriGNAI3 (Figure 1). Cells were then synchronized in mitosis and replated into fresh medium that allows
fast fork progression. In the first S phase after replating, the overall initiation
density in the AMPD2 locus dropped, so that there was a low frequency of multiple initiations occurring
in individual loci, as expected by the 'passive' mechanism. However, initiation did
not occur predominantly at the primary origin, oriGNAI3, but was distributed among the dormant origins oriA-oriF as well as oriGNAI3. Only in the second S phase after the increase in fork speed was the dominance of
oriGNAI3 regained and the relative efficiency of initiation at oriA-oriF decreased. It therefore appears that cells had adapted to growth under slow fork conditions
by raising the efficiency of oriA-oriF, which under normal conditions usually remain dormant.

Figure 1. A simplified version of the AMPD2 locus is shown, with the primary origin oriGNAI3 on the left and two less efficient origins on the right. During G1, origins are licensed
by binding Mcm2-7 (blue, M); when origins fire during S phase, Mcm2-7 provides essential
helicase activity at the fork. The cartoons on the right show the chromatin of the
locus coiled up and cross-linked to proteins of the nuclear matrix (green dots), forming
a 'halo' of DNA around the tethering points. (a) Cells adapted for growth under conditions of slow fork movement. Multiple origins
fire in the locus, with all origins having become relatively efficient (large red
ovals) to compensate for slow fork movement, and all being associated with matrix
proteins. (b) In the first cell cycle after a shift to conditions allowing fast fork progression,
the rate of origin firing is decreased, but the relative efficiency and the matrix-association
properties of the origins are similar to those seen before the shift. Only in the
second cell cycle after the shift do the two relatively inefficient origins become
dormant again (small red ovals) and less closely associated with the matrix.

Changes to chromatin loops correlate with the adaptation

Courbet et al. [1] go on to show that the adaptation of origin efficiency correlates both with changes
in chromatin organization and the association of replication origins with the nuclear
matrix in G1. Previous work has shown that during S phase, clusters of 5-50 adjacent
replicons (the stretch of DNA replicated from a given origin) are replicated together
in 'factories', with all the DNA replicated in a single factory remaining co-localized
within the nucleus over many cell cycles [11-13]. The physical basis for this organization is currently unknown. One suggestion is
that it reflects the attachment of specific DNA sequences to an insoluble nucleoskeleton
or matrix, thereby creating chromatin loops that define functional units of transcription
and replication [14]. A slightly different view is that chromatin loops may be held together by multiple
weak and reversible interactions between chromatin-bound proteins [15,16]. Whatever its physical basis, there is known to be a good correlation between the
size of DNA loops in the 'halo' of DNA that appears to be tethered to the nuclear
matrix and the average spacing between replication origins [17].

When Courbet et al. grew cells for several generations under conditions where replication forks moved
slowly, both the primary (oriGNAI3) and dormant (oriA-oriF) origins were used with similar efficiencies, and they were distributed fairly similarly
throughout the halo of matrix-attached DNA. The total size of these halos was smaller
than that of halos from cells grown under normal conditions (see cartoons on the right-hand
side of Figure 1). When cells were grown for several generations under conditions that allow fast
fork progression, the DNA halos became larger (consistent with the lower average density
of origins [17]), and oriGNAI3, but not the dormant origins, was preferentially found closer to the center of the
halo. Critically, when synchronized cells were changed from 'slow-fork' conditions
to conditions allowing normal fork rates, the change in halo size and the relative
positions of the origins within the halo were only seen in the second cell cycle after
the switch (Figure 1b).

This suggests that the positioning of origins within the halo, which correlates with
the relative efficiency of these origins, is a long-term adaptation to changes in
fork rate that persists into the next cell cycle. In some way, information about how
the origins in a cluster have been replicated is converted into a changed organization
of the nuclear matrix and the attachment of replication origins to it. Because we
do not understand how replication origins are organized within replication factories
and in chromatin loops, we can only speculate on what these changes might be. Some
marker of where forks initiate or terminate might be left on the DNA. Proteins such
as topoisomerase II [18-20] and cohesin [21,22], which are involved in the organization of DNA within the nucleus and which interact
with the replication machinery, could be involved. When such proteins are deposited
on chromatin, this might physically bring origins together with the matrix to increase
their firing efficiency.

The Mcm2-7 proteins that license replication origins are commonly misregulated at
an early stage in cancer cells [23-25], and the incorrect regulation of replication origins may be an important cause of
the genetic instability seen in cancer. The work of Courbet et al. [1] confirms the plasticity of origin usage during DNA replication and provides clues
as to how origin selection might occur in animal cells. Although this problem has
been apparent for many years, there is now promise that it can be better understood.